Systems and Methods for Effecting a Physical Change in a Biological Sample

ABSTRACT

The present invention relates generally to systems and methods for processing a biological sample that result in a physical change, such as reacting two molecules together to form a reaction product or for use in lysing viruses or biological cells for analysis using biological assay systems. As such, the present invention relates both to breaking apart biological species such as viruses and cells, as well as the formation of reactants from one or more reactive species. The sample has a volume in the range from about 1 microliter to 10 milliliters. The sample is processed by applying pressure, and either sonic energy or thermal energy to the sample, wherein the pressure achieved is usually at least 24 atmospheres, and the temperature of the sample is usually raised to at least 50° C.

The present invention relates generally to systems and methods forprocessing a biological sample that result in a physical change, such asreacting two molecules together to form a reaction product or for use inlysing viruses or biological cells for analysis using biological assaysystems. As such, the present invention relates both to breaking apartbiological species such as viruses and cells, as well as the formationof reactants from one or more reactive species.

In one embodiment of the present invention, certain biological samplescontaining cellular components must be pretreated in order to disruptthe cell wall and release intracellular components for preparation ofsamples for analysis. This treatment procedure is generally known as“lysis”, and various methods of lysing are known in the field. The lysisof robust cellular components, such as microbial spores, can bedifficult. Spores are the most difficult species to lyse because oftheir strong exterior wall, which allows them to survive extremes of theenvironment and remain viable.

One of the most common methods used to lyse robust cellular speciesinvolves the use of sonication, which causes pressure transduction, or“cavitation” of the sample. This method utilizes high frequency energywaves to break apart the walls of the cells. This is an effective methodfor partially lysing cells, but it does not always result in completelysis, solubilization and inactivation of the species in question. Onemethod for sonication is to place the sample in a single use containerwith no moving parts, and to introduce sonic waves into the container.The main disadvantages of this method are the incomplete lysis andsolubilization of the cells in the biological sample, and the generationof aerosols and bubbles. This presents a significant hazard to the user,as intact aerosolized biological agents create the greatest risk fortoxic or infectious exposure. Since there is no guarantee that the agentis fully inactivated by this simple method, it may still be hazardous tothe user.

Another problem with using sonication alone is that some cellularspecies require large amounts of energy to be introduced in order toinactivate and lyse the cells in a biological sample. The most commonexample of this is bacterial spores. The most effective way to introducethe sonic energy into the sample is by using a sonic probe, which comesinto direct contact with the sample. This is most often done in an opencontainer, for easy access to the sample. This is particularlydangerous, because as the sample is sonicated, a large portion will bereleased into the air. Also, a large amount of energy will be needed inorder to effectively complete the lysis, which may result in heating ofthe sample and inactivation of the cellular components of interest.

Accordingly, there is a need for systems and methods to pretreatbiological samples that are safe and easy to use, and produce a suitableyield of the biological components of interest.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide systems and methodsfor pretreating biological samples and particularly for lysing ofviruses and cells in biological samples or for providing the energynecessary to carry out a reaction between two chemical species or withina chemical species.

According to one aspect of the present invention, a method of effectinga physical change in a biological sample is provided, which comprisesthe steps of: placing the sample (for example, 1 microliter to 10milliliters, or 1 microliter to 1 milliliter) in a sample chamber havingat least one opening; inserting a plug into the opening to seal thechamber; applying a pressure of at least 30 psi (for example, 350 psi to500 psi) to the chamber; and applying either thermal energy (forexample, above 50° C., or 50° C. to 200° C., or 100° C. to 250° C.) orsonic energy, or both sonic energy and thermal energy, to the sample.

The sample may be known to contain or may be suspected of containingcells, subcellular structures like mitochondria, or acellular particles,such as macromolecular complexes, liposomes, vesicles, beads, or acombination thereof. In other aspects, the particle is a solid that hasa compound chemically bonded to the surface thereof.

The pressure may be varied simply by adjusting the depth to which theplug is inserted into the chamber, or the pressure may be adjusted byregulating the gaseous pressure above the sample chamber. Additionally,the sonic energy may be supplied in the form of a sonication probe or byapplying ultrasound waves. If a sonication probe is used, it may or maynot come in direct contact with the sample.

To optimize the reaction conditions during the procedure, either or boththe pressure and the temperature may be monitored and adjusted asnecessary to be increased, decreased, cycled or kept constant.

In one embodiment, the method is performed in a multi-well plate holdinga plurality of samples.

In another embodiment of the invention, the method is used for lysis,with the combination of pressure and sonication resulting in biologicalmolecules being released from cells in the sample to form a lysate. Theinitial pressure applied to the sample may be around 30 psi, and thesample may be heated to a temperature sufficient to increase thepressure to at least 24 atmospheres (350 psi).

According to another aspect of the invention, a system is provided forpracticing the method which comprises: a sample container having atleast one chamber for containing the sample, the chamber having at leastone opening; a plug adapted for sealing the opening of the chamber andfor applying pressure to the chamber; a securing device for securing theplug in the chamber.

Other aspects of the invention are described throughout thespecification

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded side elevation view of the components of thesystem according to a first embodiment of the invention;

FIG. 2 is an exploded cross-sectional view of the components on the line2-2 of FIG. 1;

FIG. 3 is a side elevational view illustrating the system of FIGS. 1 and2 assembled and ready for use;

FIG. 4 is a cross-sectional view on the line 4-4 of FIG. 3;

FIG. 5 is a side elevation view similar to FIG. 3 illustrating amodified system according to another embodiment of the invention whichallows a sonication probe to be inserted into a sample;

FIG. 6 is a cross-sectional view on the line 6-6 of FIG. 5;

FIG. 7 is a side elevation view similar to FIG. 5 illustrating asonication probe in position;

FIG. 8 is a cross-sectional view on the line 8-8 of FIG. 7;

FIG. 9 is an exploded side elevation view of the components of amulti-well lysing system according to another embodiment of theinvention;

FIG. 10 is a cross-sectional view of the multi-well and plug plates ofFIG. 9 in an assembled condition; and

FIG. 11 is a perspective view of the multi-well plate and plug plate ofthe system of FIGS. 9 and 10.

FIG. 12 is a graphical representation of the results of the Experimentdescribed below.

DETAILED DESCRIPTION OF THE INVENTION

The drawings illustrate various embodiments of a system that can be usedto effect a chemical change in a biological sample. By way of example, asystem that is useful to lyse and solubilize a biological samplecontaining robust components such as spores is depicted and describedherein. However, it should be understood that such a system is alsouseful for providing the energy necessary to effect the reaction betweenand among chemical species, such as the formation of a reaction productfrom two starting components or the breaking apart of a chemical speciesor multi-species complex, such as a liposome or subcellular structure.The disclosed technology enables researchers and other interestedparties to make lysates from cellcontaining biological samples or asolubilized mix of proteins, DNA, and any other extra or intracellularmacromolecules from variety of organism and cell types, includingviruses, bacteria (prokaryotic), bacterial spores, plant cells, andeukaryotic (mammalian) cells. The systems and methods described hereincan be used to provide extracts of proteins and nucleic acids in anamount sufficient to allow hrther characterization or experimentationwith such extracts.

As used herein, the term “biological sample” refers to material of abiological origin, which may be a body fluid, tissue sample, researchsample containing bioimolecules, and which also be from an environmentalsource, a body of water, etc., and which may optionally contain or besuspected of containing single-celled and/or multicellular organisms.Such samples may include organs, tissues, cells, spores, organelles,molecular aggregates such as hybridized nucleic acids and aggregatedproteins, single molecules, as well as portions thereof, andcombinations thereof. For example, prokaryotic cells, eukaryotic cellsand any combination thereof can be included in the biological sample,such as cells from microorganisms, animals and plants. In particular,the biological sample includes bacterial cells, bacterial spores,viruses, prions, eukaryotic cells (blood cells, tissue, white cells),bone marrow, bone, or any combination thereof.

The term “cell”, as used herein, is intended to encompass prokaryoticcells, eukaryotic cells, phage particles, and organelles.

The terms “lysate” or “cell lysate”, as used herein, refer to acomposition comprising at least some cells that have ruptured cell wallsand/or cell membranes.

As used herein, the term “lysing,” with reference to a cell suspension,refers to rupturing the cell walls and/or cell membranes, cellularcomponents, organelles of at least a portion of the cells such that atleast part of the contents, e.g. biological molecules of the cells arereleased. In certain embodiments of the method of the present invention,at least a portion of the biological material is lysed to form a lysate.Without being bound by any particular theory of operation, thebiological sample lyses under physico-chemical forces created by thecombination of the appropriate solvent environment, along with pressureand either heat or cavitation, or a combination of the two. Biologicalmolecules that are released upon lysing include nucleic acids,carbohydrates, amino acids, proteins, peptides, DNA, RNA, and anycombination thereof. Biological samples are typically aqueous, whichmeans they contain an effective amount of water molecules to cause themto be in the liquid state.

As used herein, the term “lysate” refers to the products of lysingbiological material, for example, the biological molecules that arereleased as listed above. Although most lysates will be readily solublein the biological sample fluid, certain lysate portions, such ashydrophobic components, may require additional steps to ensure at leasta portion of the lysate is solubilized. Examples of additional steps forensuring solubilization of the lysates include a suitable surfactant (ordehydrant), such as sodium dodecyl sulfate (SDS), which is typicallyincluded in the buffer, or any combination thereof. Lysatesolubilization may also be assisted using vigorous mixing, shearing,heating in surfactant, cavitation, bead beating, boiling, degassing, orany combination thereof.

“Solibulizing”, as used herein with reference to a cell suspension,refers to the ability to disaggregate either partially or fully and makecellular components soluble in the lysis buffer.

As used herein, the term “analysis” refers to a process for determiningthe identity or nature of molecular components of an organism. Incertain embodiments, the analysis identifies patterns or signatures foridentifying a biological or chemical entity.

As used herein, the term “physical change” refers to the process ofeffecting a change in the biological sample in terms of the nature andsize of its constituents, and includes without limitation, the processof lysing cells, reacting chemical species to make them smaller orlarger, breaking up chemical complexes to form smaller constituents.Accordingly, such “physical” changes include the formation of productsof a chemical reaction, as well as the deformation of constituents ofthe sample, such as cells and biological complexes.

As used herein “sonic energy’ refers to energy produced by either asonication device such as a sonication probe, or an ultrasoundtransducer capable of producing ultrasound waves.

Although the following systems and methods are described as beingapplied primarily to lyse or break open biologic species and solubilizethe proteins/nucleic acids, those skilled in the art will appreciatethat the disclosed systems and methods can be used to lyse andsolubilize substantially any type of cell component. In addition thesystem may be use as a reaction vessel to perform mixing and/or heatingat a range of temperatures to perform chemical and enzymatic reactions,synthesis of reactants form chemical monomers, such as organicchemicals, and polymers, including but not limited to DNA, proteins,alkyl hydrocarbons, inethacrylate, and acrylate polymers, and drugmoieties. This method is not limited to the bench top application, andcan be made to be useable by technicians in the field, such as fieldtechnicians investigating white powder incidents and also crime sciencefor fast processing of samples.

FIGS. 1 to 4 illustrate a lysing system 10 according to a firstembodiment of the invention. The parts of the system are shown separatedin FIGS. 1 and 2, and basically comprise a vial 12 having an internalchamber 14 for receiving a sample, the vial having an open upper end 15,a plug or insert 16 for sealing the open upper end 15 of the vial orchamber, a retaining cap 18 for holding the plug in the vial, and asonication head 19 for applying ultrasonic waves to a sample in chamber14. A heater 20 surrounds the lower end of the vial for heating thecontents of the chamber. The vial, plug and cap, when assembled as inFIGS. 3 and 4, form a tight gas and liquid sealed container, imperviousto the escape of gas and liquid under high temperature, high pressure,or both.

The plug 16 has an enlarged head 22 at its upper end for seating againstthe upper end of the vial and a reduced diameter, elongate plunger orpiston of predetermined length for extending into the chamber in orderto pressurize the contents of the chamber. A pair of spaced annulargrooves are provided on the outer surface of the plug for receivingO-ring seals 25 which seal against the inner surface of the vial as theplug is forced into the vial. The O-ring seals are made of a suitableheat resistant, resilient material such as rubber or a synthetic rubbersuch as Viton®, manufactured by Dupont De Nemours of Wilmington, Del.Although O-ring seals are illustrated in the exemplary embodiment, theseals may instead be permanently attached to the plug.

The cap 18 has internal threads 26 for threaded engagement withcorresponding external threads 28 on the outer surface of the vial tosecure the plug in the chamber. A flat inner end surface 29 of the capacts as an abutment surface which engages with the head 22 of the plugto force it into the vial as the cap is screwed onto the vial. The outersurface of the cap may be smooth or may be provided with gripping ridgesor other roughening to facilitate gripping and turning of the cap fortightening or loosening purposes.

Although the cap is secured on the vial by threaded engagement in theillustrated embodiment, the screw threads may be replaced in alternativeembodiments by any other suitable fastener arrangement, such as abayonet-type twist lock connection. The cap restricts rotation of theplug as it is pressed down into the vial, reducing wear on the O-ringseals, and assists in fully inserting the plug in the vial chamber inorder to provide the desired pressure. However, in alternativeembodiments of the invention, where rotation of the plug is not aproblem, the plug may be formed integrally with the cap as an extensionfrom the inner surface 29 of the cap.

The vial, plug and cap are preferably all of the same material to avertany gaps caused by disparate thermal expansion and contractioncoefficients. In certain embodiments, the material for the plug expandsat a greater rate than that of the vial, which increases the sealingability as the temperature increases. The material selected must also berelatively rigid and strong to maintain its integrity and shape underhigh pressure conditions. O-rings are preferable for sealing the plugsurface, to maintain a stiff plug yet have a deformable surface to matewith the vial. The material must also be selected to withstand theextreme temperature ranges common in thermal lysis. The presentlypreferred material is PEEK (Polyetheretherketon) although PC(polycarbonate), PMMA Polymethylmethacrylate), Acrylic, PDMS(Polydimethylsiloxane), and Polyolefin (ZEONOR®, Zeon Chemicals,Louisville, Ky.) are also suitable. The plug, vial and cap may be madeby injection molding.

The heater 20 in the illustrated embodiment is a coil of resistiveheating wires wrapped around the outer surface of the vial adjacent thecontained sample. Other types of heating device may be used inalternative embodiments. The heater may alternatively comprise a blockheater, RF induction heater, microwave heater, or the like.

In the illustrated embodiment, the inner surface of the vial iscylindrical and of uniform diameter along the majority of its lengthfrom the open end, with a reduced diameter portion 30 adjacent the lowerend of the vial for receiving a sample to be processed, The lower closedend 32 of the chamber is conical in shape, for increased strength underpressurized conditions, and also to make processed sample recoveryeasier. In an alternative embodiment, the interior surface of vial mayhave a tapered section. The tapered section is formed such that theinterior diameter of the tube is greatest at the open end and that thetube wall inclines to increasing thickness with increasing distance fromthe aperture. The plug will be shaped and dimensioned for mating withthe tapered section. A cooperative fit between the sealing insert andthe tapered section below the aperture aids in maintaining a tight sealbetween the sealing insert and the tube during compression.

The purpose of the plug is two-fold. First, it is designed to seal thechamber so that the sample is not lost in processing. Second, it createsand maintains pressure in the chamber during heating and/or sonication.The length of the plug is selected in order to give an initial pressureto the chamber. The length of the plug must be at least 1/2.5 of thechamber length. This will create an initial pressure of 2.5 atmospheresin the chamber. The length of the plug can be increased if greaterinitial pressures are desired, according to the following relationship:

(P1*V1)/(n1*R*T1)=(P2*V2)/(n2*R*T2)

where P is pressure, V is volume, n is the number of moles, R is aconstant, and T is temperature. R is a constant and can be eliminatedfrom both sides of the equation. Similarly we assume that the number ofmoles does not change (this is not exactly true, but the error will onlyincrease the pressure during heating). So that leaves us with:

(P1*V1)/(T1)=(P2*V2)/(T2)

In an example where the initial pressure is 1 atm and final neededpressure is 25 atm., the initial temp is 20° C. and final temp is 200°C.,

2/V1=(1 atm/25 atm)*(200° C./20° C.)=1/2.5

Alternatively, the plug can be constructed to be longer in order toreach higher pressures for other processing. The minimum plug lengthneeded for heat lysis is 1/2.5 and the maximum length is to the top ofthe sample. One reason to use a longer plug is when lysis is performedwith sonication and no heat. The pressure acts to contain the sample andreduce bubbling. The higher the pressure, the less sample that can becontained in the compressed air. The sample plug has been designed towithstand pressures up to 500 psi and this pressure can be achievedthrough any combination of mechanical force, heat, additional airvolume, or any of the three alone.

The system of FIGS. 1 to 4 is described below as used for lysing cellsin a biological sample, and the vial, plug and cap may be designed as aninexpensive single use item which is discarded after use. However, itwill be understood that the system may be used for any chemical orbiological process where a sample is to be processed using pressure,heat, and/or sonication either individually or in combination.

The vial in the illustrated embodiment is cylindrical with a cylindricalinner chamber. However, the vial and chamber cross-sections may be oval,oblong, rectangular, polygonal, or other shapes, with thecross-sectional shape of the plug matching that of the chamber forsmooth sliding engagement in the chamber.

The vial, chamber, and plug dimensions are dictated by the sample size.The sample size may be of in the range of 1 microliter up to 10milliliters in volume. It should be understood that the. systemsdescribed herein are specially adapted to a small sample size such asthis, as will be more fully explained below. A vial having a chamber ofthe order of 0.25 inches diameter and one inch length will accommodatesamples of up to 100 microliters in volume, but the range of vialchamber length may be from 0.75 inches to 4 inches, depending on desiredsample size. The plug or plunger length will be at least one half of theoverall length of the vial chamber in order to produce a 2× compressionof the air in the chamber before heat is applied. The dimensions will beselected such that the chamber dimensions when the plug 16 is fullyinserted are sufficient to hold a sample of the desired volume with thedesired gap between the upper surface of the sample and the lower end ofthe plug. The cap dimensions will be dependent on the vial outerdiameter and the cap outer diameter may be in the range of 0.25 inchesto 1.5 inches. The system can be used in a lysing method or other sampleprocessing methods according to exemplary embodiments of the invention.

The pressurization reduces the bubble development and evaporation.Bubbles are formed during boiling and also during sonication. Suchbubble formation can lead to a variety of problems in conventionalsonication systems that mostly adversely affect yield, such asaerosolation and denaturation of active biochemical species due toformation of an air-liquid interface surrounding the bubble. Byminimizing bubble formation, much smaller sample volumes (down to 1microliter) can be processed without loss of sample. Pressurization alsoreduces the amount of water vapor or sample soluble in the compressedair, which also allows for the processing of smaller sample volumes.

Suitable biological samples for processing in the method of thisinvention include a biological material in any type of solvent. Thepreferred solvent for the sample is water, which is most compatible withfurther processing steps. Almost any composition of biological materialand organic solvent can be used to form a suitable biological samplefluid. The amount of biological material in the biological sample fluidcan be as low as one spore in a fluid sample having a volume of fromabout 10 microliters to 1 ml. The weight percent of biological materialin the biological sample fluid can be as high as 100 percent, i.e. thesolvent comprising water in the biological material. Typically, theweight percent of the biological material is in the range of from about0.00000001 percent, up to about 10 percent, and preferably in the rangeof from about 0.00001 percent, up to about 1 percent. Although a varietyof additional components may be included in the biological sample fluid,for example water, salts, buffers, and contaminants, in certainembodiments the biological sample fluid consists essentially ofbiological material and a suitable solvent. In other embodiments, thebiological sample fluid consists essentially of biological material,buffer and water. In certain preferred embodiments, the biologicalsample fluid contains a high concentration of organic solvent. In otherpreferred embodiments, the biological sample contains a highconcentration of water. As used herein, the term “high concentration”refers to a composition comprising more the 30 weight percent of aspecified component based on weight of the composition.

The method may be used, for example, for lysing a cell or spore of anyorigin, e.g., prokaryotic or eukaryotic, such as bacterial cells,mammalian cells, yeast cells, insect cells, plant cells, viruses orspores. The criteria for employing conditions such as pH, buffer, ionicstrength are known to the person skilled in the art, and are essentiallythe same as those used in conventional sonication procedures.

The methods described herein can also be used to perform high throughputsynthesis of novel chemical moieties in small volumes, due to thepotential of performing low volume mixing, and high temperature heating,in the absence of boiling or bubble formation, due to the pressurizedvessel. In addition, the reaction chamber can be filled with any gas,reactive or non-reactive, oxidizing or non-oxidizing. Suitable gases forthis purpose can include but are not limited to reactive gases such asbromine, chlorine, oxygen, or inert gases such as nitrogen or helium.Other reactant or catalyzing agents, can include polymer radicalinitiators, and enzymes,

The biological samples may be derived first from other, well-knownmethods of preparing and analyzing biological materials. These methodsare capable of determining, for example, the composition of a biologicalsample in terms of protein, amino acids, DNA, mRNA, oligonucleotides,polysaccharides, or any combination thereof. Such determinations can becorrelated to a compositional database for identifying the origin of thesample, whether having a natural origin (e.g. an organism such asbacteria or virus) a, an unnatural origin (e.g. a synthetic compound orgenetically-engineering organism), or a combination of both.

The sample 33 to be lysed is placed in the bottom of the lysis chamber14. This is the recessed portion of the vial, and can handle samplevolumes as small as from 1 microliter to 0.5 mL depending on the samplevial geometry. The shape of the bottom of the vial is kept conical tomake it easy to remove the sample after processing. Conical, sphericalor tapered geometries will help to contain small sample volumes.

Once the desired sample for processing has been prepared, it is placedin the sample chamber 14 in the vial 12, and the plug or plunger 16 isinserted fully into the vial. The cap is screwed down over the upper endof the plunger to secure the plunger in place as illustrated in FIG. 4.The O-ring seals 25 create a pressure seal with the inner surface of thevial when the system is assembled as illustrated in FIG. 4. Although twoO-ring seals are used in the illustrated embodiment, a greater or lessernumber of seals may be used in alternative embodiments. The O-ring sealscan withstand pressures up to 500 psi at temperatures up to 250° C.

The sample 33 is then heated to a desired temperature. The temperaturemay be in the range from 100° C. up to 250° C., depending on the speciesto be lysed, and in an exemplary embodiment the temperature used is inthe range of 150° C. up to 200° C. As the temperature of the sample isincreased to 200° C., the pressure inside the well increases to 24 atm,or 350 psi. This increases the boiling temperature to above 220′ C, thuspreventing boiling, but does not interfere with achievement of lysis. Atthe same time, the sonication head may be activated to subject thesample to ultrasonic waves. The lysing process is completed rapidly inaround 5-10 minutes. This method provides the ability to performbio-contained sample preparation with a variety of solvents and buffercombinations, of any molecular structure.

The method employs a combination of pressure with sonication (which maycause the sample to heat up due to sonic forces) and optionally withadditional heating for lysing samples, using elevations of all threevariables in exemplary embodiments in order to lyse small samplevolumes. Various protocols may involve application of any two variables,i.e. sonication, heat, and/or pressure to achieve lysis. Application ofsonication on small sample volumes cannot be done at atmosphericpressures because the entire sample is vaporized. Pressure can becombined with temperature alone to perform the lysis. Advantageously,sonication, elevated temperature and pressure are combined to sonicatesmall volume samples. The combination of the three allows lysis to becompleted at lower temperatures than could otherwise be necessary,reducing the risk of destroying DNA strands. The increased pressure willprevent boiling of the sample and reduces creation of bubbles as aresult of the application of heat or sonication, allowing smallersamples to be processed. The sonication allows lysis to be performed ata lower temperature than would be required if heat alone were applied tothe sample, and is therefore important in the recovery of complete DNAstrands and stability. Lower temperatures allow smaller samples to beprocessed. By varying the pressure, temperature, and sonicationfrequency and amplitude, the size of the DNA fragments extracted duringlysis can be controlled.

The method of this invention is particularly useful for lysing of morerobust biologicals such as spores. Temperature alone is not enough tolyse spores or other robust species without addition of chemicals toassist in the lysis. Temperature and pressure combined, with addition ofsonication to allow the temperature to be reduced as noted above, makesit easier to lyse robust species while still retaining DNA and proteins.The temperature used will be species dependent. For vegetative cellswhich are fairly easy to lyse, the temperature may be at the lower endof the range, while it will be higher for more robust species.

In the embodiment of FIGS. 1 to 4, a sonication probe is placed againstthe lower end of the vial in order to subject the sample to ultrasonicwaves. Alternatively, in the modified lysing system of FIGS. 5 to 8, thesonication probe comes into direct contact with the sample 42. In thisembodiment, the locking cap 44 has a central opening 45 and the plug 46has a through bore 48 through which the probe 19 is inserted so that thetip 50 of the probe comes into contact with the sample 33, asillustrated in FIG. 8. Other parts of the system of FIGS. 5 to 8 areidentical to those of the previous embodiment, and like referencenumerals have been used for like parts as appropriate.

In the modified system of FIGS. 5 to 8, two separate pressure seals arerequired. The first is the double O-ring seal 25 between the plug orplunger 46 and the inner surface of the vial 12. The second is betweenthe sonication probe 19 and the passageway or through bore 48. Thesecond is between the sonication probe 19 and the passageway or throughbore 48. The sonication probe should be sealed to the plug before theplug is sealed to the bore. This seal allows the pressurization to occuras the plug is inserted, so that the sample is still contained andaerosolization is prevented. Since the sonication probe does come intocontact with the sample prior to lysing, it will need to be thoroughlydecontaminated before reuse.

The through-bore 48 may be used to add or remove sample from the vial,prior to or after insertion of the probe 19. More than one through-boremay be provided if desired; for example, for insertion of a pressuremonitor into the sample chamber. The second seal between the probeand/or temperature bore 48 may be a pressure seal such as an O-ring orthe like, or the probe may be permanently sealed in the bore with epoxyor other glue. If one or more additional through bores are provided, forexample to allow addition or removal of material, these will be sealedduring lysis. The extra fluid connections can transform the system intoa reaction vessel where multiple processes can take place. For example,the sample can first be lysed using the method described above inconnection with FIGS. 1 to 4, at the desired temperature, pressure, andsonication frequency and amplitude. After lysis is complete, additionalsolutions can be added to the lysate to prepare the sample for labeling.Finally, a reactive dye can be added to the sample to produceanalysis-ready samples. All of this can be accomplished in a discretevolume at various pressures and temperatures, and sonication can beadded to improve mixing of the constituents.

Instead of inserting the probe 19 through passageway or bore 48, itcould instead be applied externally as in the first embodiment, with thepassageway 48 either sealed during lysis or used for insertion of atemperature or pressure monitor into the sample chamber. The passagewaymay then be used as a fluid passageway for addition of other solutionsor materials to the sample after lysis, as discussed above.

FIGS. 9 to 11 illustrate a processing or lysing system 60 according toanother embodiment of the invention which can process a plurality ofdifferent samples simultaneously. The system basically comprises amulti-well plate 62 having a plurality of wells or chambers 64 each forreceiving a sample of predetermined size, a plug plate 65 having aplurality of plugs or plungers 66 projecting from one face for sealingengagement in a respective aligned well in the multi-well plate, asillustrated in FIG. 10, and a locking or latching mechanism 68 forsecuring the two plates 62 and 65 together when each plug is fullyinserted in the respective well. The locking mechanism may comprise cliplocks as indicated, or the two plates may be secured together by spacedscrew fasteners around their periphery, or by any other suitablesecuring or clamping mechanism.

In an exemplary embodiment of the invention, the multi-well plate 62 isa 96 well plate, and the plug plate has a corresponding number of plugs.The dimensions of each well and the corresponding plug are selected inthe same way as described above for the single sample system of FIGS. 1to 4, depending on the desired sample size and the desiredpressurization. The plug length is suitably sufficient to produce atleast 1/2.5 reduction in the air volume in each chamber, and the samplesizes may be of the order of 1 microliter to 1 milliliter. Each plug hasa pair of annular grooves adjacent its tip and O-ring seals 69 aremounted in the grooves for sealing engagement with the wall of the wellwhen the plug is forced into the well.

The dimensions of the 96 well plate, the layout of the wells, and thewell diameters may be the same as for a standard 96 well sample tray.This makes it easy to transfer samples from the standard tray to the 96well lyser plate, without specialized equipment. This will allow crossuse of the plug plate 95 with standard plates, providing the user withthe ability to contain and partially pressurize the sample.

A suitable heater 70 is provided for heating the sample in each well.This may be a block heater or heating coils similar to the heater 20 ofFIG. 1 around the outer surface of each well as shown by way of examplearound one of the wells in FIG. 11. An ultrasonic device 72 having aplurality of tips or heads 74 equal to the number of wells in the plateis used to apply ultrasonic waves to the sample in each well. A suitablemulti-tip ultrasonic probe for this purpose is manufactured by Misonix,Inc. of New York, such as the Misonix 96 Probe Horn.

The multi-well plate and plug plate may be made of the same material asthe single chamber vial, plug and cap. The presently preferred materialis carbon doped PEEK (Polyetheretherketon) although PC (poly carbonate),PMMA (Polymethylmethacrylate), Acrylic, PDMS (Polydimethylsiloxane), andPolyolefin (Zeonor®) are also suitable. Carbon doped PEEK can beroutinely heated to a temperature above 200″ C. This material has theadditional advantage that, when used to injection mold variouscomponents, it is bio-compatible, meaning that it does not interact withproteins or nucleic acids. Such a plate will also withstand vibration oragitation, as is produced by an ultrasonic wave generator. Standardmulti-well plates are made of polycarbonate, but this melts at lowtemperatures, not permitting the high temperatures needed for lysis ofspores. However, eukaryotic cells can be lysed at lower temperatures,so, a standard 96 well plate may be suitable for use as the plate 62 ina lysing apparatus for such materials.

The plug plate must be constructed to be sufficiently rigid to compressall of the 96 wells equally. As noted above, the plug plate and integralplugs may be injection molded from carbon doped PEEK or the like, andthe plate 64 may be thicker than the multi-well plate for addedrigidity, as seen in the drawings.

The method using the multi-chamber lysing apparatus of FIGS. 9 to 11 isessentially the same as the single vial method described above. Samplesare first placed in the wells or chambers 64, and the plug plate isaligned over the multi-well plate and lowered until each plug enters thealigned well, pressurizing the chamber. The two plates are securedtogether by latches 68 or other suitable fastener devices such asscrews. The wells are then heated and the multiprobe sonication device72 is aligned with the lower ends of the wells and moved towards thewells until each probe 74 contacts the bottom surface of the respectivealigned well, as illustrated in FIG. 10. In an alternative embodiment,the apparatus may be modified to allow the probes 72 to enter the samplechamber through the plug plate and respective plugs, in a similar mannerto the single vial apparatus of FIGS. 5 to 8. Alternatively, with theexternal sonication probe arrangement of FIG. 10, other fluidic inputsto the 96 wells may be provided through the plug plate, such as sealableconnecting conduits to allow additional materials to be supplied to therespective chambers or wells, or to allow a processed sample to beremoved From each well for further testing or processing.

The method of the present invention combines heat, sonication andpressure in order to provide novel processing capability of one or moresamples contained in sealed sample chambers. Temperature control allowschemical reaction rates to be controlled and assists in diffusing orsolvating the liquid constituents quickly. Introduction of ultrasonicwaves or sonication adds non-thermal energy which assists in thebreakdown of robust species, and also assists in the solvation ofsamples, and also can assist in mixing of materials in the sample, ifnecessary. However, prolonged sonication can cause the temperature ofthe biological sample to increase over time. Pressurization of thesample chamber prevents boiling at high temperatures and the loss of lowvolume samples. Any two of these variables can be used in combination,but the preferred method is to use all three in combination.

In the apparatus and method of the above embodiments, tight control ofthe process can be maintained which allows the optimization of sampleprocessing for a specific set of analytes which may have specificlimitations or characteristics which may require such control. Anexample is the processing of biological samples and tissues whichcontain a multiplicity of macromolecules. These individual structuralmacromolecules have a range of individual bond strength between atoms,which ‘make some molecules more or less stable under a variety ofconditions. In particular, the stability of the peptide bond in nativeproteins is considerably higher than the phosphodiester bonds of theprimary DNA sequence. As a result, the integrity of the primary aminoacid peptide bond is significantly more stable at high temperatures(greater than 150° C.) than the phosphodiester bond of DNA. Thisdifference presents significant challenge when processing such agents,such as biological spores, for analysis. In order to achieve completethermal lysis and solubilization of these agents, the optimaltemperature range (without introduction of ultrasonic energy) is between195 to 220° C. Under these conditions, the protein species remainintact, while the primary DNA structure becomes unstable and fragmentsto sizes between 0-300 bases in length. The short length of these DNAsequences makes analysis difficult. A better method is to control allelements of the reaction to prevent breakdown of DNA, whileaccomplishing the complete lysis of the sample being processed. In thisinvention, the application of heat, ultrasonic energy, and high pressureto the sample can be optimized so that lysis and solubilization ofbiological species can be completed at lower temperatures, withoutbreakdown of the DNA. This has the added advantage that the processallows for analysis of both the proteome and the genome from the samesample. This is important because the sample size available for analysisis often limited to small quantities.

The pressurization of the sample in a sealed chamber reduces theoccurrence of bubbles (which can interfere with subsequent assayprocedures) and evaporation or loss of sample. Bubbles can be formedthrough heating or boiling or as a result of vibration induced bysonication, and increasing the pressure will reduce both of theseeffects. By minimizing bubble formation and evaporation, much smallersample volumes can be processed than was possible in the past, down to 1microliter, without loss of sample. Pressurization also reduces theamount of water vapor or sample soluble in the compressed air in thechamber, which also permits processing of smaller samples. The sampleprocessing takes place in a completely bio-contained device, whichreduces the safety hazard generated by aerosolization of potentiallyhazardous biological samples.

The solubility of liquids is increased at higher pressures as are usedin the method and apparatus of this invention, so that higherconcentrations of reagents can be used during sample processing. As thepressure is increased, the sample will contain more dissolved gasparticles. Any of the sample that does evaporate is more likely torejoin the sample at higher pressures.

This system and method enables researchers and other interested partiesto make lysates or a solubilized mix of proteins, DNA, and any otherextracellular or intracellular macromolecules from variety of organismand cell types, including viruses, bacteria (prokaryotic), bacterialspores, plant cells, and eukaryotic (such as mammalian) cells. Themethod is particularly versatile when a controlled combination ofpressure, temperature, and sonication is used, and the conditions areoptimized for the particular sample to be processed. When used forlysing, the method provides extracts of proteins and nucleic acids in anamount sufficient to allow further characterization or experimentationwith such cell components.

Example Preparing Lysate From Bacillus SD. Spores

A system essentially as depicted in FIGS. 1 and 2 was used to lyse asuspension of spores from Bacillus sp. A disposable collection vial wasused for both sample containment and lysis. The vial was inserted into athermal controlled well, and the temperature was raised to 95° C. Thevial was pressurized to approximately 100 PSI, and the sample was pulsesonicated (10×, 3 second cycles, over 1 minute) at four different powerlevels (5.5 watts, 16.5 watts, 33 watts and 49.5 watts). Using a 40×objective microscope, the samples were observed to contain decreasingamounts of in-tact spores as the power was increased. At the highestpower level, the samples contained virtually no in-tact spores.

Aliquots of each sample were taken after treatment, and the DNA wasanalyzed using 260 nm wavelength to assess concentration and 280 nmwavelength to assess integrity. The results are depicted in FIG. 12.(BT=Bacillus thurengensis, and ATR=Bacillus atrophaeus.) According tothese results, an increasing amount of DNA is released as the power isincreased. Additionally, the power setting of 16.5 appeared to beoptimal for the generation of the greatest amount of in-tact DNA.

The information presented above is provided to give those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the preferred embodiments of the invention, and is notintended to limit the scope of what the inventor regards as hisinvention. Modifications of the above-described modes for carrying outthe invention that are obvious to persons of skill in the art areintended to be within the scope of the following claims. Allpublications, patents, and patent applications cited in thisspecification are incorporated herein by reference as if each suchpublication, patent or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A method of effecting a physical change in a biological sample,comprising the steps of: a) placing the sample in a sample chamberhaving at least one opening, the sample having a volume in the rangefrom about 1 microliter to 10 milliliters; b) inserting a plug into theopening of the chamber to seal the chamber; c) applying a pressure of atleast 30 psi to the chamber; and d) applying either thermal energy orsonic energy to the sample.
 2. The method as claimed in claim 1, whereinthe sample volume is in the range from 1 microliter to 1 milliliter. 3.The method as claimed in claim 1, wherein step d) further comprisesapplying sonic energy to the sample.
 4. The method as claimed in claim1, wherein step d) further comprises applying thermal energy.
 5. Themethod as claimed in claim 4, wherein the sample temperature is raisedfrom between from 50° C. to 200° C.
 6. The method as claimed in claim 1,wherein step c) further comprises applying a pressure of from 350 psi to500 psi.
 7. The method as claimed in claim 1, wherein both sonic energyand thermal energy are applied to the sample.
 8. The method as claimedin claim 1, wherein the sample contains or is suspected of containingcells.
 9. The method as claimed in claim 1, wherein the sample containsor is suspected of containing a plurality of cells or acellularparticles.
 10. The method as claimed in claim 1, wherein the pressure isvaried by adjusting the volume of the chamber in which the sample issealed.
 11. The method as claimed in claim 1, wherein the pressure isvaried by varying the depth of insertion of the plug into the chamber.12. The method as claimed in claim 1, wherein a plurality of samples areplaced into respective individual sample chambers in a multi-well plate,a plurality of plugs on a single plug plate are inserted into the openends of the respective chambers so as to seal and pressurize thechambers.
 13. A method of effecting a physical change in a biologicalsample, comprising the steps of a) placing the sample in a chamberhaving at least one opening, the sample having a volume in the rangefrom about 1 microliter to 10 milliliters; b) inserting a pressurizationplug into the opening far enough to apply an initial pressure of atleast two atmospheres to the chamber; and c) subjecting the sample tosonic energy.
 14. The method as claimed in claim 13, further comprisingthe step of increasing the pressure in the chamber to at least 350 psi.15. The method as claimed in claim 13, further comprising the step ofheating the sample to a temperature in the range from 100° C. to 250° C.16. The method as claimed in claim 13, wherein the step of subjectingthe sample to sonic energy comprises inserting a sonication probethrough the plug into the chamber until the probe directly contacts thesample.
 17. The method as claimed in claim 13, wherein the step ofsubjecting the sample to sonic energy comprises applying a sonicationprobe externally to the chamber wall.
 18. The method as claimed in claim14, further comprising the step of monitoring the pressure within thechamber throughout step c).
 19. The method as claimed in claim 15,further comprising the step of monitoring the temperature within thechamber throughout step c).
 20. A system for effecting a physical changein a biological sample, comprising: a) a sample container having atleast one chamber for containing the sample, the chamber having at leastone opening; b) a plug adapted for sealing the opening of the chamberand for applying pressure to the chamber; c) a securing device forsecuring the plug in the chamber, and d) a sonication device forsubjecting a sample in the chamber to sonication.
 21. The system asclaimed in claim 35, wherein the plug has at least one seal for sealingengagement with the chamber when inserted into the opening.
 22. Thesystem as claimed in claim 21, wherein the seals comprise O-ringsmounted on the plug.
 23. The system as claimed in claim 20, wherein thechamber has a predetermined length and the plug has a length equal toabout half the length of the chamber.
 24. The system as claimed in claim20, wherein the securing device comprises a cap, the cap and the samplecontainer having interengageable formations for releasably locking thecap over the opening and securing the plug in the chamber.
 25. Thesystem as claimed in claim 24, wherein the interengageable formationscomprise external threads on the sample container adjacent the openingand internal threads on the cap for releasable threaded engagement withthe external threads on the sample container.
 26. The system as claimedin claim 20, wherein the plug has at least one through bore and thesecuring device has an opening aligned with the through bore when theplug is inserted into the opening to provide access to the chamber. 27.The system as claimed in claim 26, further comprising a device insertedthrough the opening and through bore into the chamber, the device beingselected from the group consisting of: the sonication device, a pressuresensor, a temperature sensor, and a fluid flow tube for supplyingmaterial to the sample chamber or removing material from the samplechamber.
 28. The system as claimed in claim 20, wherein the sonicationdevice comprises an external probe with an end secured in contact withthe chamber opposite the opening.
 29. The system as claimed in claim 20,wherein the plug has a through bore and the sonication device has aprobe for insertion through the through bore and into the sample in thechamber, a seal being formed between the probe and the through bore. 30.The system as claimed in claim 20, further comprising a heater mountedadjacent the chamber for heating the sample to a temperature between100° C. to 250° C.
 31. The system as claimed in claim 20, furthercomprising a temperature sensor for monitoring temperature in thechamber, and a controller for selectively varying the temperature. 32.The system as claimed in claim 20, further comprising a pressure sensorfor monitoring pressure in the chamber.
 33. The system as claimed inclaim 20, wherein the sample chamber further comprises a reduceddiameter portion opposite the opening for containing the sample.
 34. Thesystem as claimed in claim 20, wherein the chamber has a diameter ofapproximately 0.2 to 0.3 inches and a length in the range from 0.7 to 4inches.
 35. The system as claimed in claim 34 wherein the plug has alength of at least half the length of the chamber.
 36. The system asclaimed in claim 20 wherein the chamber and plug are made of the samematerial.
 37. The system as claimed in claim 36, wherein the material isselected from the group consisting of: polyetheretherketone,polycarbonate, polymethylmethacrylate, acrylic, polydimethylsiloxane,and polyolefin.
 38. The system as claimed in claim 37, wherein thematerial is polyetheretherketone.
 39. The system as claimed in claim 20,wherein the sample container further comprises a multi-well plate havinga plurality of sample chambers, and wherein the system further comprisesa plurality of plugs.
 40. The system as claimed in claim 39, furthercomprising a cover plate for covering the openings of the samplechambers, the plugs being formed integrally with the cover plate andprojecting from the cover plate for sealing engagement in the openingsof respectively aligned sample chambers when the cover plate is securedover the multi-well plate.
 41. The system as claimed in claim 40,wherein the securing device comprises a suitable fastener mechanism forsecuring the cover plate to the multi-well plate with the plugsextending into and pressurizing the respectively aligned samplechambers.
 42. The system as claimed in claim 41, wherein each plug hasat least one seal for sealing engagement with the sample chamber. 43.The system as claimed in claim 42, further comprising a heater assemblyfor heating samples in the chambers substantially uniformly.